Analytical instruments such as biosensors are well established as a means of recording the progress of biomolecular interactions in real time. Biosensors employ a variety of transduction technologies to detect interactions between biomolecules. Such instrumentation requires microfluidic channels in order to deliver samples to a sensing region, and pumps and valves are preferred means for moving sample through the channels in a controlled reproducible manner.
Recent interest in microfluidics technology has come about because of a growing need for sophisticated control of fluid streams for such sensing applications. A number of prior systems, referred to as integrated microfluidic cards, are composed of a series of substantially planar substrates possessing channels and structures that when bonded form internal passages and active components such as valves and pumps. Despite much progress these systems are rarely as robust as conventional flow injection analysis fluidic systems where the active components are not integrated into the fluidic card; however, these non-integrated systems typically have relatively large dead volumes.
There are several transducers capable of recording the progress of the biomolecular interactions to be detected. One example is a quartz crystal microbalance. Binding of molecules to the surface of a quartz crystal changes the fundamental resonance frequency which allows quantification of the binding event. Other technologies include light scattering, reflectometric interference spectroscopy, ellipsometry, fluorescence spectroscopy, calorimetry, and evanescent field based optical detection. A particularly effective evanescent field based technology, known as surface plasmon resonance (SPR), exploits the behavior of light upon reflection from a gold-coated optical substrate, for example.
SPR is an optical technique that enables real-time monitoring of changes in the refractive index of a thin film close to the sensing surface where materials to be tested are located (typical material types include a ligand attached to the sensing surface, a fluid buffer, and a fluid analyte which is contained (e.g. soluble or insoluble colloidal solution) in a running/flowing material that is to bind with the ligand and be tested). The evanescent field created at the surface decays exponentially from the surface and falls to one third of its maximum intensity at approximately 300 nanometers (nm) from the surface. Hence the SPR technique is sensitive to surface refractive index changes.
The delivery of samples to the SPR active sensing regions is made possible by creating flow channels that cover the active sensing regions. Each flow channel possesses an inlet and outlet to allow for the flow of buffer or samples over the SPR active sensing regions. The thin film sensing surface is derivatized to possess a polymeric coating that enables biomolecules (“ligands”) to be permanently immobilized on the coating. The immobilized biomolecules usually possess binding specificity for another biomolecule contained in the sample (the “analyte”). The strength of this binding is given by the affinity constant which is simply the ratio of the binding rate constant divided by the dissociation rate constant. It is possible to measure these constants because an SPR-based biosensor records the progress of binding and dissociation events in real time.
Of particular interest is the kinetic analysis of such interactions. Important constants to measure include the association and dissociation between analyte in the flowing sample and ligands immobilized onto the sensing area. Other factors (e.g., mass transport characteristics of the sample) are also important to know. These are determined by flowing liquid mixtures containing one or more compounds (the analyte(s)) over sensing areas on which receptor chemicals (the ligand(s)) are located to interact with reactive contents of the mixtures. Using different concentrations of samples (i.e., different amounts of the one or more analytes in the mixtures) is important to enable such characteristics to be measured accurately.
There are different ways of achieving multiple concentration information from these known types of systems. One conventional way is to use different injectable volumes of solutions, each having a different concentration. One solution is flowed across the sensing area and data collected. The sensing area is then regenerated by flowing a “cleansing” fluid over the sensing area to remove the previous sample's contents. That is, usually when an analyte binds to an immobilized ligand the interaction must be reversed (“regenerated”) in order to run another analyte injection. However, regenerating the surface can damage some of the immobilized ligand thereby resulting in decreased surface capacity for a subsequent test.
Sequentially injected volumes of solutions with different concentrations can be used without regeneration as disclosed in WO 2004/109284. In this step injection approach, the analyte is injected in a first volume at a low concentration then followed by a higher concentration in another volume and so forth until the surface is saturated. In this way it is possible to obtain data representing binding at different concentrations without using regeneration. These different concentrations also can be obtained from a mixture having a varying concentration within itself. See, also, WO 2004/109295, US 2003/0143565, and “Nonregeneration protocol for surface plasmon resonance: Study of high affinity interaction with high density biosensors.” Tang, Y., Mernaugh, R. and Zeng, X. (2006), Anal. Chem., 78, 1841-1848.
According to “Analyte gradient-surface plasmon resonance: A one-step method for determining kinetic rates and macromolecular binding affinities.” Shank-Retzlaff, M. L. and Sligar, S. G. (2000), Anal. Chem., 72, 4212-4220, regeneration of a surface can be avoided by continuously injecting a sample in which a concentration gradient exists due to operation of an in-line gradient maker. The kinetic models used for processing the data to determine the desired kinetic constants are modified to account for the changing analyte concentration during the single injection.